Two important molecular signatures of a human cell's response to DNA damage are the phosphorylation of a histone variant H2AX and a change in the transcriptional program. Recent results from our laboratory have implicated the RVB1 protein in the cellular response to DNA damage. Human RVB is an integral part of a chromatin remodeling complex containing the histone acetyltransferase, TIP60, the ATPase p400 and several other cellular proteins. RVB1 or TIP60 depletion leads to an increase in phosphoH2AX accumulation after DNA damage and sensitizes cancer cells to DNA damaging agents, suggesting that inhibitors of RVB ATPase may be useful in chemo- or radiotherapy. This project will test two hypotheses: 1) RVB1 is required to neutralize the inhibitory action of p400 on the acetyltransferase activity of the TIP60 complex and 2) the acetyltransferase activity of TIP60 is required to mobilize phosphoH2AX containing nucleosomes off the DNA prior to its dephosphorylation by cellular phosphatases. Aim 1 will explore whether TIP60 is the primary chromatin remodeler involved in down modulating phosphoH2AX and whether RVB2 co-operates with RVB1 in this function. Aim 2 will test whether the hypothesis of TIP60 regulation by RVB1 and p400 is applicable to chromatin remodeling at promoters controlled by TIP60. Aim 3 will test the role of ATP binding/hydrolysis and oligomerization with self or with RVB2. In vitro assays will be developed to explore the molecular basis of how RVB1 activates the TIP60 acetyltransferase complex and how the TIP60 complex mobilizes phosphoH2AX-containing nucleosomes prior to dephosphorylation of phosphoH2AX. PUBLIC HEALTH RELEVANCE The cellular response to DNA damage determines both how environmental DNA damaging agents lead to mutations and cause cancers and how therapeutic DNA damaging agents treat cancers. Chromatin remodelers regulated by RVB act both at sites of DNA damage and at promoters to control the cell's response. Therefore a molecular understanding of how RVB regulates chromatin remodeling factors important for the optimal response to DNA damage will allow the design of chemicals that interfere with RVB function and thus sensitize cells to chemo- or radio-therapy.